STING mediates lupus via the activation of conventional dendritic cell maturation and plasmacytoid dendritic cell differentiation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): B Cell, Dendritic Cell, Blood
DISEASE(S): Wounds And Injuries
SUBMITTER: 
Poorichaya Somparn
LAB HEAD: Poorichaya Somparn
PROVIDER: PXD019239 | Pride | 2020-09-14
REPOSITORIES: Pride
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Project description:The pathogenesis of rheumatoid arthritis (RA) is complicated and involves both innate and adaptive immunity [5]. The innate immunity such as macrophages or fibroblast-like synoviocytes (FLS) in the synovium can generate inflammatory mediators, including TNF-α, IL-1, IL-6, IL-17, IFN-γ, and chemo kines that lead to synovial inflammation, bone erosion, and cartilage damage [6-8]. The IL-17 signaling mediates the autoantibody production in collagen-induced arthritis (CIA) model [9]. However, the treatment response with an anti-IL17 monoclonal antibody in RA patients shows a high degree of heterogeneity [10]. The inhibitors of the Janus kinase (JAK) pathway are approved for RA patients [11]. These data suggest that the targeted multiple cytokines through the JAK pathway are useful for RA treatment. Disturbance of type I IFNs (IFNs-I) signaling and production drive autoimmune development [12]. The presymptomatic RA patients display an increase of IFNs-I before the onset of symptoms [13]. The RA patients also show the elevation of IFN- α in the synovial fluid and high expression of IFNs-I regulated gene in peripheral blood mononuclear cells (PBMC) [14]. However, the role of IFNs-I in arthritis and bone homeostasis has suggested the accelerating effect of arthritis and bone damage. The interferon-alpha receptor knockout mice develop arthritis severity higher than wild-type mice in the model of antigen-induced arthritis [15]. IFNs-I also affects the bone homeostasis by inhibiting osteoclastogenesis via receptor activator of nuclear factor-kappa B (RANK) pathway, and reduction of c-FOS expression [16-18]. Therefore, the goal of RA treatment with antagonizing the IFNs-I pathway has to be optimized between efficacy and potentially adverse effect. STING is a cytosolic DNA sensor that initiates the production of IFNs-I. STING functions have been reported as both pro-inflammatory signaling and negative regulator against inflammation [19-21]. The mutation in exon 5 of the STING gene results in gain function leading to initiate inflammation and cause the Sting associated vasculopathy with onset in infancy (SAVI) [22]. Loss of STING function rescues DNaseII-deficient mice from lethality and polyarthritis [23]. However, Sting-deficient lupus mice (MRL/Lpr mice) show higher and earlier mortality than Sting-sufficient MRL/Lpr mice [24]. The pathology also shows lymphoid hypertrophy with a higher amount of macrophages and granulocytes infiltration, autoantibodies, and cytokine [24]. The objective of this study was to identify the role of STING in the pathogenesis of rheumatoid arthritis using collagen-induced arthritis (CIA) model as a representative model of the human RA.
2020-06-26 | PXD018652 | Pride
Project description:The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5- methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of chromatin structure and gene expression. Isolation and sorting was performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield, Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. GC nuclei from WT and Mecp2-null mice were sorted by selecting a specific FSC/SSC population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in GC EGFP+ animals resulted >92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us to enrich the sample from 65-70% to 92-96% on GC cells. 5hmC was pulled down as described (Song et al., 2010). After purification DNA was amplified as described in TruSeq DNA Sample kit. 1-0.5 M-NM-<g of DNA was used for each experiment. Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina, PEM-bM-^@M-^P 102M-bM-^@M-^P 1003).), following by amplification with Illumina primers. Input samples were produced for each cell types in both procedures 5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36- bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best). Two biological replicas were done for each of the cell types Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA from cerebellum were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) wit
2013-01-14 | E-GEOD-42880 | biostudies-arrayexpress
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5 An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
2010-06-20 | E-GEOD-18834 | biostudies-arrayexpress
Project description:gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences).
2019-10-15 | GSE134101 | GEO
Project description:High cell density Matrigel beads (HDB) were printinto onto large skin wounds (10 mm and 15 mm). Compared with acellular beads printing, HDB group could achieve more efficient injury regeneration.
2022-10-12 | GSE214868 | GEO
Project description:994820 Fear conditioning: Top2B immunoprecipitated from mouse hippocampus dissected 30 minutes after Fear Conditioning
994821 Control: Top2B immunoprecipitated from hippocampus dissected from mouse that were in home cages.
Complete description:
Hippocampus brain tissue (4 mouse per sample) from were lysed in in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% N-Lauroylsarcosine, phosphatase and protease inhibitors cocktail) by passing through a 25-gauge needle and incubated with Benzonase Nuclease (>=250 units/microliters) 2microliters for 0.3ml of sample for 3hrs followed by sonication. Samples were centrifuged at 20000 x g for 5 min. The supernatants were collected, and protein concentrations determined using the BCA assay. Equal amounts of protein and 6 microliters of Top2B antibody (Rabbit, Abcam, ab125297) per sample were incubated overnight. Samples were incubated with 25 microliters of protein A/G-magnetic beads for 3 h and the beads were washed 4 times with high salt buffer (500 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS) and one more time with TBS with 0.1% Triton X-100. All procedures were performed at 4C. Beads were re-suspended in Laemmli buffer to separate proteins by electrophoresis.The gel was stained with Coomassie blue and the Topoisomerase 2 band was cut.
2022-07-06 | MSV000089825 | MassIVE
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:Proteasome inhibitor bortezomib (BTZ) induces apoptosis in myeloma (MM) cells, and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine MM models, we here show that BTZ also triggers immunogenic cell death (ICD) characterized by exposure of calreticulin (CALR) on dying MM cells, phagocytosis of tumor cells by dendritic cells, and induction of MM specific immunity. We identify a BTZ-triggered specific ICD-gene signature which confers improved outcome in two independent MM patient cohorts. Importantly, BTZ stimulates MM cells immunogenicity via activation of cGAS/STING pathway and production of type-I interferons; and STING agonists significantly potentiate BTZ-induced ICD. Our studies therefore delineate mechanisms whereby BTZ exerts immunotherapeutic activity, and provide the framework for clinical trials of STING agonists with BTZ to induce potent tumor-specific immunity and improve patient outcome in MM.
2021-04-12 | GSE171837 | GEO
Project description:The modified iCLIP protocol is based on the previously described protocol (Huppertz et al., 2014) with modifications that enable the definition of readthrough cDNAs. HeLa cells were crosslinked with 0.15mJ/cm2 254nm UV light. Protein G Dynabeads were used for immunoprecipitations (IP). For each IP, 100 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCL pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and incubated with the anti-eIF4A3 antibody or polyclonal mAb BB7 serum anti-PTBP1. To prepare the cell lysate, the cells were lysed with 1 mL iCLIP lysis buffer (final concentration 2mg/mL), sonicated (Bioruptor, 5x5 sec on/off), incubated with RNase I (1-2 x10-3 U/mL for eIF4A3) at 37C for 3 min, and centrifuged. After the pellet had dissolved, the mixture was diluted with H2O to 1000 L and an additional centrifugation was performed. The supernatant of the iCLIP lysis buffer (1 mL) and the MSB lysis buffer (1 ml) were combined and added to the antibody-coupled beads, which were then rotated at 4C for 2 h (final urea concentration 175 mM). The beads were then washed with high-salt washing buffer (50mM Tris-HCL pH 7.4, 1M NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). After the first round of washes, the library was split into 10%, which were radioactively labelled (according to the basic iCLIP protocol), and 90%, which proceed through 3 adapter addition, an additional phosphorylation (0.2 l PNK, 0.4 l cold ATP (1mM), 0.4 l 10x PNK buffer, 3 l water) and a 5 marker ligation (6 l water, 5 l 4X ligation buffer, 2 l RNA ligase, 1 l RNasin, 2 l 5 marker (100 M), 4 l PEG400). The sequence of the 5 marker is CAGUCCGACGAUC, which corresponds to the Illumina short RNA 5 Adapter (RA5), part #15013205; this sequence is not complementary to the primers used for amplification of iCLIP cDNA libraries (Huppertz et al., 2014). We then produced sequence reads of 120 nt using the Illumina HiSeq platform for eIF4A3 iCLIP. The rest of the protocol was carried out as previously described (Huppertz et al., 2014). Huppertz, I., Attig, J., D'Ambrogio, A., Easton, L.E., Sibley, C.R., Sugimoto, Y., Tajnik, M., Konig, J., and Ule, J. (2014). iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287.
2016-11-24 | E-MTAB-4000 | biostudies-arrayexpress